An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs

Take minced murine lung tissue in a digestion buffer and incubate at a physiological temperature.

Buffer enzymes break down the tissue matrix, loosening the cells.

Triturate to dissociate the cellular mass, and filter the suspension using a strainer to isolate the single cells.

Centrifuge to remove the supernatant with tissue debris. Incubate the cells with a lysis buffer to lyse the red blood cells.

Centrifuge to discard the cellular debris-containing supernatant. Resuspend the cells and transfer them to a microplate.

Add antibodies to block the Fc receptors, preventing background staining.

Introduce a fluorophore-labeled antibody cocktail targeting specific surface markers on the innate immune cells — monocytes, macrophages, dendritic cells, natural killer cells, neutrophils, eosinophils, and adaptive immune cells — B and T lymphocytes.

Fix and permeabilize the cells. Add fluorophore-labeled antibodies specific for an intracellular marker on monocytes, macrophages, dendritic cells, and eosinophils.

Using flow cytometry, classify the labeled immune cells.

Transfer the lung to a Petri dish. Mince it using two fine scalpels, and then place it into a 50-milliliter conical tube. Add 5